1. Field of the Invention
The present invention relates to a confocal observation system, and more particularly, to a confocal observation system such as a microscope or an endoscope including scanning optical systems.
2. Description of the Related Art
The confocal scanning microscope is a microscope which two-dimensionally scans a sample by means of light such as that of a laser beam or the like, detects, by an optical detector, the reflected light, the transmitted light or the fluorescence from the sample via an optical system including an objective lens, and obtains two-dimensional intensity information of the reflected light, the transmitted light or the fluorescence.
Also, in the confocal scanning microscope, the fluorescence image of the sample can be observed by displaying the above intensity information as a two-dimensional distribution of the intensity on a CRT or the like. The confocal scanning microscope such as above suppresses the scatted light which does not come from the measured point by combining a point light source such as a laser beam light source and a pinhole and by irradiating the sample with pin point accuracy. Also, by using the pinhole as a spatial filter provided in front of the optical detector which images the light existing on the plane including the measured point around the pinhole, the light from the plane which has been shifted to the optical axis direction is prevented from passing through the pinhole.
According to the above method, an optically sliced image can be obtained.
Also, in the confocal scanning microscope, the sample is two-dimensionally scanned by a spot beam and the focal point is moved by a prescribed pitch in the Z axis direction which is the optical axis direction so that a plurality of the sliced images are obtained, accordingly, sliced images of a three-dimensional space (referred to as a Z-stack image, hereinafter) can be obtained as a result.
Additionally, when multiphoton absorption is caused by using a component which emits pulsed laser beam or the like as the laser light source, only the portion of the specimen lying in the focal plane will be excited. Accordingly, even when the pinhole is not provided, only information about the focal plane of the specimen will be obtained.
Also, when another scanning optical system is provided in addition to the optical system for acquiring the image, the irradiation by the laser beam for photostimulation and the acquisition of the image can be conducted separately.
When three-dimensional information of the biological sample is to be obtained by the confocal scanning microscope such as that above, the three-dimensionally scanned region is searched and determined by moving a stage which carries the sample while monitoring the two-dimensional image displayed on a CRT or the like.
As a method of determining the three-dimensionally scanned region, a technique is disclosed in Japanese Patent Application Publication No. 2003-195174. In this technique, an example in which dendritic information of a cell is obtained is employed. First, the laser beam irradiates the 3-D sample and a Z-stack image of the sample is obtained.
Next, by using at least two of the two-dimensional data made by reflecting the obtained Z-stack image onto the XY plane, the two-dimensional data made by reflecting the obtained three-dimensional image onto the YZ plane and the two-dimensional data made by reflecting the obtained three-dimensional image onto the ZX plane, the curve data for the basic path of the nerve cell or the like for example included in the sample is obtained from the two-dimensional data.
By scanning the sample in accordance with the curve data for the basic path, the three-dimensional information of the nerve cell or the like in the sample can be obtained.
Also, in the document of U.S. Pat. No. 6,855,941 B1, a technique for a multiphoton excitation laser microscope which requires adjustment for preparing suitable observation conditions, in which the simple setting of conditions is realized by the arrangement of the optical members is disclosed. Also, in the document of U.S. Pat. No. 6,094,300, a method in which another scanning optical system in addition to the detecting optical system is provided so that the photostimulation is caused separately from the observation position is proposed.